Oligonucleotide modulation of protein kinase C

ABSTRACT

Compositions and methods are provided for the treatment and diagnosis of diseases associated with protein kinase C. Oligonucleotides are provided which are specifically hybridizable with a PKC gene or mRNA. Oligonucleotides specifically hybridizable with a particular PKC isozyme, set of isozymes or mRNA transcript are provided. Methods of treating conditions amenable to therapeutic intervention by modulating protein kinase C expression with an oligonucleotide specifically hybridizable with a PKC gene or mRNA are disclosed. Compositions and methods are provided for the treatment, detection and diagnosis of diseases associated with protein kinase C alpha and specific transcripts thereof. New nucleic acid sequences are provided which encode 3&#39; untranslated regions of human protein kinase C alpha polynucleotide probes for PKC alpha are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the national phase of international Ser. No.PCT/US94/07770 filed Jul. 8, 1994, which is a continuation of Ser. No.08/199,779 filed Feb. 22, 1994, now U.S. Pat. No. 5,681,747 issued Oct.28, 1997, which is a continuation of Ser. No. 08/089,996 filed Jul. 9,1993, now U.S. Pat. No. 5,703,054 issued Dec. 20, 1997, which is acontinuation in part of Ser. No. 07/852,852 filed Mar. 16, 1992.

FIELD OF THE INVENTION

This invention relates to therapies, diagnostics, and research reagentsfor disease states which respond to modulation of the expression ofprotein kinase C. In particular, this invention relates to antisenseoligonucleotides specifically hybridizable with nucleic acids relatingto protein kinase C. These oligonucleotides have been found to modulatethe expression of protein kinase C. Palliation and therapeutic effectresult.

BACKGROUND OF THE INVENTION

The phosphorylation of proteins plays a key role in the transduction ofextracellular signals into the cell. The enzymes, called kinases, whicheffect such phosphorylations are targets for the action of growthfactors, hormones, and other agents involved in cellular metabolism,proliferation and differentiation. One of the major signal transductionpathways involves the enzyme protein kinase C (PKC), which is known tohave a critical influence on cell proliferation and differentiation. PKCis activated by diacylglycerols (DAGs), which are metabolites releasedin signal transduction.

Interest in PKC was stimulated by the finding that PKC is the major, andperhaps only, cellular receptor through which a class of tumor-promotingagents called phorbol esters exert their pleiotropic effects on cells[Gescher et al., Anti-Cancer Drug Design 4:93-105 (1989)]. Phorbolscapable of tumor production can mimic the effect of DAG in activatingPKC, suggesting that these tumor promoters act through PKC and thatactivation of this enzyme is at least partially responsible for theresulting tumorigenesis [Parker et al., Science 233:853-866 (1986)].

Experimental evidence indicates that PKC plays a role in growth controlin colon cancer. It is believed that specific bacteria in the intestinaltract convert lipids to DAG, thus activating PKC and altering cellproliferation. This may explain the correlation between high dietary fatand colon cancer [Weinstein, Cancer Res. (Suppl.) 51:5080s-5085s(1991)]. It has also been demonstrated that a greater proportion of thePKC in the colonic mucosa of patients with colorectal cancer is in anactivated state compared to that of patients without cancer [Sakanoue etal., Int. J. Cancer 48:803-806 (1991)].

Increased tumorigenicity is also correlated with overexpression of PKCin cultured cells inoculated into nude mice. A mutant form of PKCinduces highly malignant tumor cells with increased metastaticpotential.

Sphingosine and related inhibitors of PKC activity have been shown toinhibit tumor cell growth and radiation-induced transformation in vivo[Endo et al., Cancer Research 51:1613-1618 (1991); Borek et al., Proc.Natl. Acad. Sci. 88:1953-1957 (1991)]. A number of experimental orclinically useful anti-cancer drugs show modulatory effects on PKC.Therefore, inhibitors of PKC may be important cancer-preventive ortherapeutic agents. PKC has been suggested as a plausible target formore rational design of conventional anti-cancer drugs [Gescher, A. andDale, I.L., Anti-Cancer Drug Design, 4:93-105 (1989)].

Experiments also indicate that PKC plays an important role in thepathophysiology of hyperproliferative skin disorders such as psoriasisand skin cancer. Psoriasis is characterized by inflammation,hyperproliferation of the epidermis and decreased differentiation ofcells. Various studies indicate a role for PKC in causing thesesymptoms. PKC stimulation in cultured keratinocytes can be shown tocause hyperproliferation. Inflammation can be induced by phorbol estersand is regulated by PKC. DAG is implicated in the involvement of PKC indermatological diseases, and is formed to an increased extent inpsoriatic lesions.

Inhibitors of PKC have been shown to have both antiproliferative andantiinflammatory effects in vitro. Some antipsoriasis drugs, such ascyclosporine A and anthralin, have been shown to inhibit PKC. Inhibitionof PKC has been suggested as a therapeutic approach to the treatment ofpsoriasis [Hegemann, L. and G. Mahrle, Pharmacology of the Skin, H.Mukhtar, ed., p. 357-368, CRC Press, Boca Raton, Fla., 1992].

PKC is not a single enzyme, but a family of enzymes. At the present timeat least seven isoforms (isozymes) of PKC have been identified: α, β, γ,δ, ε, ζ and η. These isozymes have distinct patterns of tissue and organlocalization (see Nishizuka, Nature, 334:661-665 (1988) for review) andmay serve different physiological functions. For example, PKC-γ seems tobe expressed only in the central nervous system. PKC-α and -β areexpressed in most tissues, but have different patterns of expression indifferent cell types. For example, both PKC-α and PKC-β are expressedin, and have been purified from, human epidermis. While PKC-α has beendetected mainly in keratinocytes of the basal layers of the epidermis,PKC-β is found mainly in the middle layers of the epidermis andLangerhans cells. PKC-η has been found predominantly in the skin andlungs, with levels of expression much higher in these tissues than inthe brain. This is in contrast to other members of the PKC family whichtend to be most abundantly expressed in the brain [Osada et al., J.Biol. Chem. 265:22434-22440 (1990)]. Another PKC isozyme, PKC-η, isbelieved to play a critical role in control of proliferative cascades.This was demonstrated by using antisense RNA, peptide inhibitors or a15-mer phosphorothioate antisense oligonucleotide targeted to the AUG ofXenopus PKC-η to deplete PKC-η levels in Xenopus oocytes. These depletedoocytes were shown to be resistant to maturation in response to insulin,while the maturation pathway activated by progesterone was not affected.WO 93/20101. While the PKC isozymes listed here are preferred fortargeting by the present invention, other isozymes of PKC are alsocomprehended by the present invention.

It is presently believed that different PKC isozymes may be involved invarious disease processes depending on the organ or tissue in which theyare expressed. For example, in psoriatic lesions there is an alterationin the ratio between PKC-α and PKC-β, with preferential loss of PKC-βcompared to normal skin [Hegemann, L. and G. Mahrle, Pharmacology of theSkin, H. Mukhtar, ed., p. 357-368, CRC Press, Boca Raton, Fla., 1992].

Even for a given isozyme, there may be multiple RNA transcriptsexpressed from a single gene. In the case of PKCα, for example, two mRNAtranscripts are seen: a long (approximately 8.5 kb) transcript and ashort (approximately 4 kb) transcript. Multiple PKCα transcripts areproduced from the murine and the bovine PKCα genes as well. The ratiobetween the long and short transcripts varies between species and isbelieved to vary between tissues as well. In addition, there may be somecorrelation between this ratio and the proliferative state of cells.

Although numerous compounds have been identified as PKC inhibitors (seeHidaka and Hagiwara, Trends in Pharm. Sci. 8:162-164 (1987) for review),few have been found which inhibit PKC specifically. While the quinolinesulfonamide derivatives such as1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) inhibit PKC atmicromolar concentrations, they exhibit similar enzyme inhibitionkinetics for PKC and the CAMP-dependent and cGMP-dependent proteinkinases. Staurosporine, an alkaloid product of Streptomyces sp., and itsanalogs, are the most potent in vitro inhibitors of PKC identified todate. However, they exhibit only limited selectivity among differentprotein kinases [Gescher, Anti-Cancer Drug Design 4:93-105 (1989)].Certain ceramides and sphingosine derivatives have been shown to havePKC inhibitory activity and to have promise for therapeutic uses,however, there remains a long-felt need for specific inhibitors of theenzymes.

There is also a desire to inhibit specific PKC isozymes, both as aresearch tool and as treatment for diseases which may be associated withparticular isozymes. Godson et al. [J. Biol. Chem. 268:11946-11950(1993)] recently disclosed use of stable transfection of antisense PKC-αcDNA in cytomegalovirus promotor-based expression vectors tospecifically decrease expression of PKC-α protein by approximately 70%.It was demonstrated that this inhibition causes a loss of phospholipaseA₂ -mediated arachidonic acid release in response to the phorbol esterPMA. Attempts by the same researchers at inhibiting PKC activity witholigodeoxynucleotides were ultimately unsuccessful due to degradation ofoligonucleotides.

OBJECTS OF THE INVENTION

It is a principal object of the invention to provide therapies forneoplastic, hyperproliferative, inflammatory and other disease statesassociated with protein kinase C.

Another object of the invention is to provide selective therapies fordiseases associated with particular isozymes of protein kinase C.

It is a further object of the invention to provide antisenseoligonucleotides which are capable of modulating the expression ofprotein kinase C.

Another object of the invention is to provide antisense oligonucleotideswhich are capable of selectively modulating the expression of particularisozymes of protein kinase C.

Yet another object is to provide means for diagnosis of diseasesassociated with protein kinase C.

A further object of the invention is to provide means for differentialdiagnosis of diseases associated with particular isozymes of proteinkinase C.

A still further object of the invention is to provide research tools forthe study of the effects of protein kinase C expression and diseasesassociated therewith.

An additional object of the invention is to provide research tools forthe study of the effects of expression of particular isozymes of proteinkinase C and diseases associated therewith.

It is an object of the invention to provide novel nucleic acid moleculesencoding a 3'-untranslated region of human PKCα, including sequencesunique to the long mRNA transcript of PKCα.

Another object of the invention is to provide antisense oligonucleotideswhich are capable of selectively modulating the expression of particularmRNA transcripts of PKCα.

A further object of the invention is to provide polynucleotide probesfor detection of human PKC.

A still further object of the invention is to provide polynucleotideprobes for detection of particular mRNA transcripts of PKCα.

A further object of the invention is to provide means for differentialdiagnosis of diseases associated with particular mRNA transcripts ofPKCα.

It is an object of the invention to provide therapies for neoplastic,hyperproliferative, inflammatory and other disease states associatedwith PKCα.

Another object of the invention is to provide selective therapies fordiseases associated with particular mRNA transcripts of PKCα.

An additional object of the invention is to provide research tools forthe study of the effects of expression of particular transcripts of PKCαand diseases associated therewith.

These and other objects of this invention will become apparent from areview of the instant specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(a) and 1(b) are graphical depictions of the effects on PKCexpression of antisense oligonucleotides hybridizable with PKC-α.Oligonucleotides are arranged by PKC target region, 5' to 3'.

FIG. 2 is a line graph showing dose-dependent reduction of PKC-α proteinlevels after oligonucleotide treatment of A549 cells. ♡=ISIS 4632CCCCAACCACCTCTTGCTCC; SEQ ID NO:120; ▪=ISIS 4649 SEQ ID NO:3; =ISIS4636 GTTCTCGCTGGTGAGTTTCA; SEQ ID NO:121; ▴=ISIS 4648GAGACCCTGACCAGTTGATC; SEQ ID NO:122.

FIG. 3 is a bar graph showing reduction of PKC-α MRNA after treatment ofA549 cells with oligonucleotides. Hatched bars represent the 8.5 kbtranscript, plain bars represent the 4.0 kb transcript. The followingoligonucleotides were used: ISIS-3520 (SEQ ID NO:1), ISIS-3521 (SEQ IDNO:2), ISIS-3522 (SEQ ID NO:3), and ISIS-3527 (SEQ ID NO:5).

FIG. 4 is a line graph showing the relationship between deoxy gap lengthand activity of chimeric oligonucleotides against PKC.

FIG. 5 is a line graph showing dose response curves for chimericoligonucleotides (all SEQ ID NO: 3) with different deoxy gap lengths.

FIG. 6 is a bar graph showing the effects of several 2'--O--methylchimeric oligonucleotides of SEQ ID NO: 3 on PKC-α mRNA levels. Hatchedbars represent the 8.5 kb transcript, plain bars represent the 4.0 kbtranscript.

FIG. 7 is a bar graph and diagram showing the effects of several2'--O--methyl and 2'--O--propyl chimeric oligonucleotides (6996, 7273)of SEQ ID NO: 3 on PKC-α mRNA levels. Hatched bars represent the 8.5 kbtranscript, plain bars represent the 4.0 kb transcript.

FIG. 8 is a bar graph and diagram showing the effects of additional2'--O--methyl and 2'--O--propyl chimeric oligonucleotides (7008, 7294)of SEQ ID NO: 3 on PKC-α mRNA levels. Hatched bars represent the 8.5 kbtranscript, plain bars represent the 4.0 kb transcript.

FIGS. 9A and 9B are a set of bar graphs showing the effect of additionaloligonucleotides on PKC-α mRNA levels. FIG. 9A shows oligonucleotides6632 (SEQ ID NO:52), 6653 (SEQ ID NO:52) and 6665. FIG. 9B showsoligonucleotides 3521 (for comparison SEQ ID NO:2), 7082 (SEQ ID NO:53),7083 (SEQ ID NO:53) and 7084 (SEQ ID NO:53). Hatched bars represent the8.5 kb transcript, plain bars represent the 4.0 kb transcript.

FIG. 10 is a line graph showing anti-tumor activity of ISIS 3521 (SEQ IDNO:2). Each dashed line represents tumor volume in one animal treatedwith control oligonucleotide; each solid line represents tumor volume inone animal treated with ISIS 3521.

FIGS. 11A and 11B are a set of line graphs showing effect ofoligonucleotides on growth of human MDA-MB231 tumors in nude mice. FIG.11A shows results obtained with ISIS 3521 (SEQ ID NO:2); FIG. 11B showsresults obtained with ISIS3527 (SEQ ID NO:5). Each line represents tumorvolume in one animal. =control; ∘=oligonucleotide at 60 mg/kg;Δ=oligonucleotide at 6 mg/kg.

FIG. 12 is a bar graph showing effect of 20-mer phosphorothioateoligonucleotides on PKC-η expression in A549 cells. The followingoligonucleotides were used: 6432 (SEQ ID NO:43), 6433 (SEQ ID NO:44),6443 (SEQ ID NO:42), 6431 (SEQ ID NO:40), 6442 (SEQ ID NO:41), 6441 (SEQID NO:46), 6435 (SEQ ID NO:45), 6436 (SEQ ID NO:54), 6434 (SEQ IDNO:55), 6444 (SEQ ID NO:56), 6445 (SEQ ID NO:57), 6446 (SEQ ID NO:58),6553 (SEQ ID NO:59), 6581 (SEQ ID NO:47), 6605 (SEQ ID NO:60), 6580 (SEQID NO:48), 6579 (SEQ ID NO:61), and 6603 (SEQ ID NO:62).

FIG. 13 is a nucleotide sequence (SEQ ID NO: 104) of a portion of the 3'untranslated region of the human PKCα gene beginning at the Bcl I sitenear the 3' end of the previously known sequence and extending in the 3'direction. Newly determined sequences begin at nucleotide 56 and areunderlined (SEQ ID NO:105). Bold sequences are unique to the long mRNAtranscript of PKCα (SEQ ID NO:106).

FIG. 14 is a line graph showing a time course of PKCα mRNA levels incells (shown as percent of control) after treatment with oligonucleotide7911 (SEQ ID NO: 117). Levels of both the short and long mRNAtranscripts are indicated. Levels of short mRNA transcript arerepresented by solid lines. Levels of long mRNA transcript arerepresented by dotted lines. By 12 hours after treatment with ISIS 7911(SEQ ID NO: 117), levels of both messages were reduced by over 80%.

SUMMARY OF THE INVENTION

In accordance with the present invention, oligonucleotides are providedthat are specifically hybridizable with DNA or RNA deriving from thegene that encodes PKC. The oligonucleotide comprises nucleotide unitssufficient in identity and number to effect such specific hybridization.This relationship is commonly denominated as "antisense". In onepreferred embodiment, the oligonucleotides are specifically hybridizablewith the translation initiation codon of the gene, and preferablycomprise a sequence CAT. In another preferred embodiment, theoligonucleotides are specifically hybridizable with the 5'-untranslatedor 3'-untranslated regions of the gene. In yet another preferredembodiment, oligonucleotides are provided that are specificallyhybridizable with DNA or mRNA encoding a particular PKC isozyme or aparticular set of PKC isozymes. Such oligonucleotides may beconveniently and desirably presented in a pharmaceutically acceptablecarrier.

In accordance with other preferred embodiments, the oligonucleotidescomprise one or more chemical modifications which convey some desiredcharacteristic such as improved target affinity, cellular uptake orstability in the presence of cellular nucleases. Examples ofmodifications having such utility are 2'--O--alkyl and 2'-fluoro sugarmodifications and phosphorothioate backbone modifications.

Other aspects of the invention are directed to methods for modulatingthe expression of PKC or of a particular PKC isozyme or set of isozymesin cells or tissues. Additional aspects of the invention are directed tomethods of detection in cells or tissues of the DNA or RNA that encodesPKC and specific detection in cells or tissues of RNA or DNA thatencodes particular PKC isozymes. Such methods comprise contacting cellsor tissues suspected of containing said gene with oligonucleotides inaccordance with the invention in order to interfere with the effect ofor to detect said RNA or DNA.

Other aspects of the invention are directed to methods for diagnosticsand therapeutics of animals suspected of having a disease associatedwith PKC or one of its isozymes. Such methods comprise contacting theanimal or cells or tissues or a bodily fluid from the animal witholigonucleotides in accordance with the invention in order to modulatethe expression of PKC, to treat conditions associated with PKC, or toeffect a diagnosis thereof.

This invention provides nucleic acid sequences that encode portions ofthe 3' untranslated region of human PKCα. Polynucleotide probes andmethods of detecting PKCα are also provided. In some embodiments of thepresent invention, nucleic acid sequences specific for a particular mRNAtranscript of PKCα are provided, as well as polynucleotide probes andmethods for specific detection of this transcript.

In accordance with other embodiments of the present invention, antisenseoligonucleotides are provided that are specifically hybridizable withnucleic acids encoding PKCα. In still other embodiments, antisenseoligonucleotides are provided which are specifically hybridizable with aparticular mRNA transcript of PKCα. Such oligonucleotides may beconveniently and desirably presented in a pharmaceutically acceptablecarrier.

In accordance with still other aspects of the invention are providedmethods for modulating the expression of PKCα or of a particular PKCαmRNA transcript in cells. Additional aspects of the invention aredirected to methods of detection in cells of nucleic acids that encodePKCα and specific detection in cells of nucleic acids that encodeparticular PKCα transcripts. Such methods comprise contacting the cellswith oligonucleotides in accordance with the invention in order tointerfere with the effect of or to detect said nucleic acid.

In still other embodiments of the invention are provided methods fortreating animals having a disease associated with expression of PKCα orone of its transcripts. Such methods comprise contacting the animal witha therapeutically effective amount of oligonucleotides in accordancewith the invention in order to modulate the expression of PKCα, to treatconditions associated with PKCα, or to effect a diagnosis thereof.

DETAILED DESCRIPTION OF THE INVENTION

Antisense oligonucleotides are now accepted as therapeutic agents havingpromise for the treatment of many human diseases. Oligonucleotidesspecifically bind (hybridize) to the complementary sequence of DNA,pre-mRNA or mature mRNA, as defined by Watson-Crick base pairing,interfering with the flow of genetic information from DNA to protein.The properties of antisense oligonucleotides which make them specificfor their target sequence also make them extraordinarily versatile.Because antisense oligonucleotides are long chains of monomeric units,they may be readily synthesized for any target RNA sequence.

Numerous recent studies have documented the utility of antisenseoligonucleotides as biochemical tools for studying target proteins(Rothenberg et al., J. Natl. Cancer Inst., 81:1539-1544 (1989); Zon, G.,Pharmaceutical Res., 5:539-549 (1988). Because of recent advances inoligonucleotide chemistry and synthesis of oligonucleotides whichexhibit enhanced cell uptake, target binding affinity and nucleaseresistance, it is now possible to consider the use of antisenseoligonucleotides as a novel form of therapeutics. For example, antisenseoligonucleotides targeted to c-myb have been used to completelyeliminate myeloid leukemia cells from bone marrow derived from patientswith acute myelogenous leukemia. Gewirtz and Calabretta, U.S. Pat. No.5,098,890. An antisense oligonucleotide has been shown to have clinicalefficacy in humans for treatment of cytomegalovirus retinitisinfections.

Antisense oligonucleotides offer an ideal solution to the problemsencountered in prior art approaches to the treatment of conditionsassociated with PKC. They can be designed to selectively inhibit a givenisozyme or particular set of isozymes, or to inhibit all members of agiven family of isozymes.

Current agents which modulate the activity or metabolism of proteinkinase C exhibit many unacceptable side effects due to their lack ofspecificity, or they exhibit only limited effectiveness in inhibitingthe enzyme. The instant invention circumvents problems encountered byprior workers by modulating the production of the enzyme, rather thaninhibiting the enzyme directly, to achieve the therapeutic effect. Inthe instant invention, the oligonucleotide is designed to hybridizedirectly to mRNA or to a gene, ultimately modulating the amount of PKCprotein made from the gene. "Hybridization," in the context of thisinvention, means hydrogen bonding, also known as Watson-Crick basepairing, between complementary bases, usually on opposite nucleic acidstrands or two regions of a nucleic acid strand, to form adouble-stranded duplex. Guanine and cytosine are examples ofcomplementary bases which are known to form three hydrogen bonds betweenthem. Adenine and thymine are examples of complementary bases which areknown to form two hydrogen bonds between them. "Specificallyhybridizable" and "substantially complementary" are terms which indicatea sufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide (or polynucleotide probe) to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays and therapeutictreatment, or, in the case of in vitro assays, under conditions in whichthe assays are conducted. It is understood that an oligonucleotide orpolynucleotide probe need not be 100% complementary to its targetnucleic acid sequence to be specifically hybridizable.

The relationship between an oligonucleotide and its complementary (or"target") nucleic acid is commonly denoted as "antisense."

It is preferred to target specific genes for antisense attack. It hasbeen discovered that the genes coding for PKC α, β, γ, δ, ε, ζ and η areparticularly useful for this approach. Inhibition of PKC expression isexpected to be useful for the treatment of diseases, particularlyhyperproliferative and inflammatory disorders.

However, "modulation" in the context of this invention means either anincrease or decrease (stimulation or inhibition) of PKC expression.

In the context of this invention, the term "oligonucleotide" refers to apolynucleotide formed from naturally occurring nucleobases andpentofuranosyl (sugar) groups joined by native phosphodiester bonds.This term effectively refers to naturally occurring species or syntheticspecies formed from naturally occurring subunits or their closehomologs.

The term "oligonucleotide" may also refer to moieties which functionsimilarly to naturally occurring oligonucleotides but which havenon-naturally occurring portions. Thus, oligonucleotides may havealtered sugar moieties, nucleobases or inter-sugar ("backbone")linkages. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake, enhanced target binding affinity and increasedstability in the presence of nucleases.

Specific examples of some preferred oligonucleotides envisioned for thisinvention are those which contain intersugar backbone linkages such asphosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages. Most preferred are those with CH₂ --NH--O--CH₂, CH₂--N(CH₃)--O--CH₂, CH₂ --O--N(CH₃)--CH₂, CH₂ --N(CH₃)--N(CH₃)--CH₂ andO--N(CH₃)--CH₂ --CH₂ backbones (where phosphodiester is O--P--O--CH₂).Phosphorothioates are also most preferred. Also preferred areoligonucleotides having morpholino backbone structures. Summerton, J. E.and Weller, D. D., U.S. Pat. No. 5,034,506. In other preferredembodiments, such as the peptide nucleic acid (PNA--referred to by someas "protein nucleic acid") backbone, the phosphodiester backbone of theoligonucleotide may be replaced with a polyamide backbone whereinnucleosidic bases are bound directly or indirectly to aza nitrogen atomsor methylene groups in the polyamide backbone see, e.g., P. E. Nielsen,M. Egholm, R. H. Berg, O. Buchardt, Science 1991, 254, 1497 and U.S.patent application Ser. No. 08/054,363, filed Apr. 26, 1993 andincorporated herein by reference. In accordance with other preferredembodiments, the phosphodiester bonds are substituted with structureswhich are chiral and enantiomerically specific. Persons of ordinaryskill in the art will be able to select other linkages for use inpractice of the invention.

Oligonucleotides may also include species which include at least onemodified nucleobase. Thus, purines and pyrimidines other than thosenormally found in nature may be so employed. Similarly, modifications onthe pentofuranosyl portion of the nucleotide subunits may also beeffected, as long as the essential tenets of this invention are adheredto. Examples of such modifications are 2'--O--alkyl-- and2'-halogen-substituted nucleotides. Some specific examples ofmodifications at the 2' position of sugar moieties which are useful inthe present invention are OH, SH, SCH₃, F, OCN, O(CH₂)_(n) NH₂ orO(CH₂)_(n) CH₃ where n is from 1 to about 10; C₁ to C₁₀ lower alkyl,substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; OF₃ ; OCF₃ ;O--, S--, or N-alkyl; O--, S--, or N-alkenyl; SOCH₃ ; SO₂ CH₃ ; ONO₂ ;NO₂ ; N₃ ; NH₂ ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino;polyalkylamino; substituted silyl; an RNA cleaving group; a reportergroup; an intercalator; a group for improving the pharmacokineticproperties of an oligonucleotide; or a group for improving thepharmacodynamic properties of an oligonucleotide and other substituentshaving similar properties. One or more pentofuranosyl groups may bereplaced by another sugar, by a sugar mimic such as cyclobutyl or byanother moiety which takes the place of the sugar.

Chimeric or "gapped" oligonucleotides are also preferred embodiments ofthe invention. These oligonucleotides contain two or more chemicallydistinct regions, each comprising at least one nucleotide. Typically,one or more region comprises modified nucleotides that confer one ormore beneficial properties, for example, increased nuclease resistance,increased uptake into cells or increased binding affinity for the RNAtarget. One or more unmodified or differently modified regions retainthe ability to direct Rnase H cleavage. Chimeric oligonucleotides aredisclosed in PCT application US92/11339 which is assigned to theassignee of the instant application and which is incorporated byreference herein in its entirety. Examples of chimeric oligonucleotideswhich are presently preferred are 2'--O--methyl or 2'--O--propyloligonucleotides having a "deoxy gap" region of 2'-deoxynucleotides.Usually this deoxy gap region is located between the two 2'-alkylregions. In these preferred embodiments, the internucleotide (backbone)linkages may be uniformly phosphorothioate or some combination ofphosphorothioate and phosphodiester linkages.

All such oligonucleotides are best described as being functionallyinterchangeable with natural oligonucleotides (or synthesizedoligonucleotides along natural lines), but having one or moredifferences from natural structure. All such oligonucleotides arecomprehended by this invention so long as they function effectively tohybridize with the PKC RNA.

The oligonucleotides in accordance with this invention preferablycomprise from about 5 to about 50 nucleotide units. It is more preferredthat such oligonucleotides comprise from about 8 to 30 nucleotide units,and still more preferred to have from about 12 to 25 nucleotide units.As will be appreciated, a nucleotide unit is a base-sugar combination(or a combination of analogous structures) suitably bound to an adjacentnucleotide unit through phosphodiester or other bonds forming a backbonestructure.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of the routineer. It is also well known to usesimilar techniques to prepare other oligonucleotides such asphosphorothioates or alkylated derivatives. Other modified andsubstituted oligomers can be similarly synthesized.

In accordance with this invention, persons of ordinary skill in the artwill understand that messenger RNA includes not only the coding region,which contains information to encode a protein using the three lettergenetic code, but also associated ribonucleotides which form a regionknown to such persons as the 5'-untranslated region, the 3'-untranslatedregion, the 5' cap region and intron/exon junction ribonucleotides.Thus, oligonucleotides may be formulated in accordance with thisinvention which are targeted wholly or in part to these associatedribonucleotides as well as to the coding ribonucleotides. In preferredembodiments, the oligonucleotide is specifically hybridizable with atranscription initiation site, a translation initiation site, a 5' capregion, an intron/exon junction, coding sequences or sequences in the5'- or 3'-untranslated region.

The oligonucleotides of this invention are designed to be hybridizablewith the PKC gene or with messenger RNA derived from the PKC gene. Suchhybridization, when accomplished, interferes with the normal roles ofthe messenger RNA to cause a modulation of its function in the cell. Thefunctions of messenger RNA to be interfered with may include all vitalfunctions such as translocation of the RNA to the site for proteintranslation, actual translation of protein from the RNA, splicing of theRNA to yield one or more mRNA species, and possibly even independentcatalytic activity which may be engaged in by the RNA. The overalleffect of such interference with the RNA function is to modulateexpression of the PKC gene.

The oligonucleotides of this invention can be used in diagnostics,therapeutics, prophylaxis, and as research reagents and kits. Since theoligonucleotides of this invention hybridize to the PKC gene and itsmRNA, sandwich and other assays can easily be constructed to exploitthis fact. Furthermore, since the oligonucleotides of this inventionhybridize specifically to particular isozymes of the PKC mRNA, suchassays can be devised for screening of cells and tissues for particularPKC isozymes. Such assays can be utilized for diagnosis of diseasesassociated with various PKC forms. Provision of means for detectinghybridization of oligonucleotide with the PKC gene can routinely beaccomplished. Such provision may include enzyme conjugation,radiolabelling or any other suitable detection systems. Kits fordetecting the presence or absence of PKC may also be prepared.

For therapeutic or prophylactic treatment, oligonucleotides areadministered in accordance with this invention. Oligonucleotides may beformulated in a pharmaceutical composition, which may include carriers,thickeners, diluents, buffers, preservatives, surface active agents andthe like in addition to the oligonucleotide. Pharmaceutical compositionsmay also include one or more active ingredients such as antimicrobialagents, antiinflammatory agents, anesthetics, and the like in additionto oligonucleotides.

The pharmaceutical composition may be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration may be done topically (includingophthalmically, vaginally, rectally, intranasally), orally, byinhalation, or parenterally, for example by intravenous drip orsubcutaneous, intraperitoneal or intramuscular injection.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condomsmay also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives.

Dosing is dependent on severity and responsiveness of the condition tobe treated, but will normally be one or more doses per day, with courseof treatment lasting from several days to several months or until a cureis effected or a diminution of disease state is achieved. Persons ofordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates.

The present invention also provides a nucleic acid molecule having asequence which encodes the 3'-untranslated region of human PKCα isprovided (FIG. 13). This sequence was determined from cDNA clonesprepared from human A549 cells, beginning with a clone overlapping the3'-most end of the previously published PKCα sequence [Finkenzeller etal., Nucl. Acids Res. 18:2183 (1990); Genbank accession number X52479]and extending in the 3' direction. A polyadenylation site which wasreached after 1080 nucleotides (nucleotide 1136 in FIG. 13); has beenidentified as the 3' end of the short (4 kb) mRNA transcript of PKCα. Anadditional 676 nucleotides of sequence in the 3' direction weredetermined, which sequence is unique to the long (8 kb) mRNA transcriptof PKCα. The nucleic acid molecule of the present invention maypreferrably be comprised of deoxyribonucleic acids and may bedouble-stranded in some aspects of the present invention. Also inaccordance with the present invention, said nucleic acid molecules areisolated. "Isolated" as the term is used herein, in meant to refer tomolecules which have been purified or synthesized so as to besubstantially homogenous. The term does not exclude the possibility thatcertain impurities may be present in the composition, but is, instead,meant to refer to the absence of non-relevant nucleic acid sequences.

In accordance with the present invention polynucleotide probesspecifically hybridizable to a portion of the 3' untranslated region ofthe human PKCα gene are provided. Polynucleotide probes specificallyhybridizable to a portion of the long mRNA transcript of PKCα are alsoprovided. Such probes may be used for diagnostic or research purposes todetect or quantitate the expression of PKCα. Probes may be used tospecifically detect or quantitate the long transcript of PKCα. Saidpolynucleotide probes may range in length from about 5 to about 50nucleotide units. In more preferred embodiments of the present inventionthe probes may be from about 8 to about 30 nucleotide units in length.Ideally, said probes range in length from about 12 to about 25nucleotide units. It is recognized that since polynucleotide probes ofthe present invention ideally do not exceed 50 nucleotides in length,said probes may specifically hybridize to only a portion of the targetedsequence. The portion of the PKCα sequence to be targeted can beidentified by one skilled in the art. Most suitably, a target sequenceis chosen which is unique, thereby decreasing background noiseattributable to hybridization by the probe other than to the target. Byway of example, one skilled in the art would be unlikely to select arepeating sequence of adenine nucleotide units as this is a commonsequence occurring in many genes. The practitioner might choose toperform a search and comparison of sequences found in a sequencedepository such as Genbank in order to identify and design a usefulprobe. Such methods are conventionally used to identify uniquesequences. These unique sequences, when used as probes, need notnecessarily be crucial to the regulation of the expression of PKCα.

The following examples illustrate the present invention and are notintended to limit the same.

EXAMPLES Example 1 Oligonucleotide Synthesis

Unmodified DNA oligonucleotides were synthesized on an automated DNAsynthesizer (Applied Biosystems model 380B) using standardphosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl-phosphoramidites were purchased from AppliedBiosystems (Foster City, Calif.). For phosphorothioate oligonucleotides,the standard oxidation bottle was replaced by a 0.2 M solution of3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwisethiation of the phosphite linkages. The thiation cycle wait step wasincreased to 68 seconds and was followed by the capping step.

2'--O--methyl phosphorothioate oligonucleotides were synthesizedaccording to the procedures set forth above substituting 2'--O--methylβ-cyanoethyldiisopropyl phosphoramidites (Chemgenes, Needham, Mass.) forstandard phosphoramidites and increasing the wait cycle after the pulsedelivery of tetrazole and base to 360 seconds. Similarly, 2'--O--propylphosphorothioate oligonucleotides may be prepared by slightmodifications of this procedure.

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides were purified by precipitation twiceout of 0.5 M NaCl with 2.5 volumes ethanol. Analytical gelelectrophoresis was accomplished in 20% acrylamide, 8 M urea, 45 mMTris-borate buffer, Ph 7.0.

The oligonucleotides tested are presented in Table 1. Sequence data arefrom the cDNA sequence published by Finkenzeller et al., Nucl. AcidsRes. 18:2183 (1990); Genbank accession number X52479. The sequencenumbers given under the oligonucleotides are relative to the firstresidue to be sequenced on the cDNA, which is 28 residues upstream ofthe ATG start codon.

                  TABLE 1                                                         ______________________________________                                        OLIGONUCLEOTIDES TARGETED TO HUMAN PKC-α                                SEQ                                                                           ID  Sequence             Target      ISIS #                                   ______________________________________                                        1   CCC CAA CCA CCT CTT GCT CC                                                                         5'          3520                                          19                  Untranslated                                         2   GTT CTC GCT GGT GAG TTT CA                                                                         3'          3521                                          2063                Untranslated                                         3   AAA ACG TCA GCC ATG GTC CC                                                                         Translation 3522                                          41                  init. codon                                          4   GGA TTC ACT TCC ACT GCG GG                                                                         3'          3526                                          2109                Untranslated                                         5   GAG ACC CTG AAC AGT TGA TC                                                                         3'          3527                                          2211                Untranslated                                         6   CCC GGG AAA ACG TCA GCC AT                                                                         Translation 3674                                          47                  init codon                                           7   CTG CCT CAG CGC CCC TTT GC                                                                         Internal    3682                                          110                 (C1) domain                                          8   AGT CGG TGC AGT GGC TGG AG                                                                         Internal    3686                                          193                 (C1) domain                                          9   GCA GAG GCT GGG GAC ATT GA                                                                         Internal    3687                                          480                 (C1) domain                                          10  GGG CTG GGG AGG TGT TTG TT                                                                         3'          3695                                           2080               Untranslated                                         11  CAC TGC GGG GAG GGC TGG GG                                                                         3'          3875                                           2098               Untranslated                                         12  AGC CGT GGC CTT AAA ATT TT                                                                         3'          3878                                           2137               Untranslated                                         13  ATT TTC AGG CCT CCA TAT GG                                                                         3'          3879                                           2168               Untranslated                                         14  AAG AGA GAG ACC CTG AAC AG                                                                         3'          3884                                           2217               Untranslated                                         15  GAT AAT GTT CTT GGT TGT AA                                                                         3'          3885                                           2235               Untranslated                                         16  ATG GGG TGC ACA AAC TGG GG                                                                         Internal    3886                                           2027               (C3) domain                                          17  GTC AGC CAT GGT CCC CCC CC                                                                         Translation 3890                                           36                 init. codon                                          18  CGC CGT GGA GTC GTT GCC CG                                                                         Internal    3891                                           63                 (V1) domain                                          19  TCA AAT GGA GGC TGC CCG GC                                                                         Internal    3892                                           1643               (C3) domain                                          20  TGG AAT CAG ACA CAA GCC GT                                                                         3'          3947                                           2151               Untranslated                                         ______________________________________                                    

Example 2 Cell Culture and Treatment with Phorbol Esters andOligonucleotides Targeted to PKC-α

PKC protein half-lives have been reported to vary from 6.7 hours to over24 hours [Young et al., Biochem. J. 244:775-779 (1987); Ballester etal., J. Biol. Chem. 260:15194-15199 (1985)]. These long half-lives makeinhibiting steady-state levels of PKC-α an unwieldy approach whenscreening antisense oligonucleotides, due to the long incubation timeswhich would be required. We have therefore made use of the ability ofphorbol esters to reversibly lower intracellular levels of PKC.Treatment of cells with phorbol esters causes an initial activation ofkinase activity, followed by a down-regulation of PKC. For PKC-α thisdown-regulation has been shown to be a direct consequence of anincreased rate of proteolysis of the kinase with no apparent change insynthetic rate.

We determined that in human lung carcinoma (A549) cells, treatment withthe phorbol ester 12,13-dibutyrate (PDBu), using a modification of themethod of Krug et al., [Krug et al., J. Biol. Chem. 262:11852-11856(1987)] lowered cellular levels of PKC-α, without affecting PKC-α mRNAlevels, and that this effect was reversible. The basis of the assay toscreen for potency of oligonucleotides targeting PKC-α is to initiallylower PKC-α protein levels by chronic treatment with PDBu, remove PDBuby extensively washing the cells (hence allowing the cells to synthesizefresh PKC-α protein), and incubate the cells with oligonucleotidesintended to inhibit the resynthesis of new PKC-α protein.

Procedure: A549 cells (obtained from the American Type CultureCollection, Bethesda, Md.) were grown to confluence in 6-well plates(Falcon Labware, Lincoln Park, N.J.) in Dulbecco's modified Eagle'smedium (DME) containing 1 g glucose/liter and 10% fetal calf serum (FCS,Irvine Scientific, Santa Ana, Calif.).

Cells were treated with 500 nM PDBu (Sigma Chem. Co., St. Louis, Mo.)for 12-16 hours (overnight). Cells were then washed three times in DMEat 37° C., and 1 ml DMA containing 20 μl DOTMA (Lipofectin reagent, BRL,Bethesda, Md.) was added. Oligonucleotides were added to a concentrationof 1 μM and the cells were incubated for a further 4 hours at 37° C.

Cells were washed once in 3 ml DME containing 0.1 mg/ml BSA and afurther 2 ml DME containing 0.1 mg/ml BSA was added. Oligonucleotides (1μM) were added and the cells were incubated at 37° C. for 24 hours.

Cells were washed three times in phosphate-buffered saline (PBS) andcellular proteins were extracted in 120 μl sample buffer (60 mM Tris pH6.8, 2% SDS, 10% glycerol, 10 mM dithiothreitol) and boiled for 5minutes. Intracellular levels of PKC-α protein were determined byimmunoblotting.

Example 3 Immunoblot Assay for PKC Expression

Cell extracts were electrophoresed on 10% SDS-PAGE mini-gels. Theresolved proteins were transferred to Immobilon-P membrane (Millipore,Bedford Mass.) by electrophoretic transfer and the membrane was blockedfor 60 minutes in TBS (Tris-HCl pH 7.4, 150 mM NaCl) containing 5%nonfat milk. The membrane was then incubated for 16 hours at 4° C. withmonoclonal antibodies raised against PKC-α (UBI, Lake Placid N.Y.)diluted to 0.2 μg/ml in TBS containing 0.2% nonfat milk. This wasfollowed by three washes in TBS plus 0.2% nonfat milk. The membrane wasthen incubated for one hour with ¹²⁵ I-labelled goat anti-mousesecondary antibody (ICN Radiochemicals, Irvine Calif.). Membranes werethen washed extensively in TBS plus 0.2% nonfat milk. Bands werevisualized and quantitated using a Phosphorimager (Molecular Dynamics,Sunnyvale, Calif.). PKC-α appears as a single band with a molecularweight of 80 kD.

Each oligonucleotide was tested three times, in triplicate, and theresults of the experiments were normalized against percentage of proteinpresent as compared to cells which were not treated with oligonucleotide(FIGS. 1a and 1b). The five most effective oligonucleotides target theAUG start codon and regions slightly upstream and downstream from it(Sequence Nos. 1, 3, 17, 7, 6). The next most effectiveoligo-nucleotides are targeted toward the 3' untranslated region of theRNA (oligos 2, 5, 14).

Example 4 Other Isozymes of PKC

Results with oligonucleotides targeting human PKC-α demonstrated thatthe most effective target sequences were those surrounding thetranslation initiation codon and the 3' untranslated region. It isbelieved that these sequences will also be effective targets foroligo-nucleotides directed against other isozymes of PKC. Antisenseoligonucleotides which are likely to be effective inhibitors of PKC areidentified below. These oligonucleotides are synthesized as in Example1, and can be screened as in Examples 2 and 3, using appropriateantibodies where available. Alternatively, a reporter gene assay systemcan be established, transiently co-expressing the desired isozyme of PKCwith luciferase under the influence of the TPA-responsive enhancer orother suitable promoter. PKC expression is then assayed by measuringluciferase activity using standard procedures. Luciferase is extractedfrom cells by lysis with the detergent Triton X-100, as described byGreenberg, M. E., in Current Protocols in Molecular Biology, (F. M.Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. A. Smith, J. G.Seidman and K. Strahl, eds.), John Wiley and Sons, NY (1987). A DynatechML1000 luminometer is used to measure peak luminescence upon addition ofluciferin (Sigma) to 625 μM.

PKC-β, Types I and II

Sequence data are from Kubo et al., FEBS Lett. 223: 138-142 (1987);Genbank accession numbers X06318, M27545, X07109. Sequences are numberedfrom the first 5' base sequenced on the cDNA. PKC-β types I and II arethe result of alternative mRNA splicing of a single gene product. Thisresults in proteins with identical amino termini (5' end of the mRNA);however, there is sequence divergence in the carboxy termini (3' end ofthe mRNA). The following oligonucleotides, targeted to the translationinitiation codon, are expected to modulate expression of both PKC-βtypes I and II:

                  TABLE 2                                                         ______________________________________                                        OLIGONUCLEOTIDES TARGETED TO PKC-β TYPES I AND II                        SEQ ID                                                                              Sequence            Target                                              ______________________________________                                        21    CAT CTT GCG CGC GGG GAG CC                                                                        Translation init.                                           139                    120                                            22    TGC GCG CGG GGA GCC GGA GC                                                                        "                                                           134                    115                                            23    CGA GAG GTG CCG GCC CCG GG                                                                        "                                                           113                     94                                            24    CTC TCC TCG CCC TCG CTC GG                                                                        "                                                           183                    164                                            ______________________________________                                    

The following antisense oligonucleotides are targeted to the3'-untranslated region of PKC-β type I:

                  TABLE 3                                                         ______________________________________                                        OLIGONUCLEOTIDES TARGETED TO PKC-β TYPE I                                SEQ. ID                                                                              Sequence            Target                                             ______________________________________                                        25     TGG AGT TTG CAT TCA CCT AC                                                                        3' Untranslated                                             2168                  2149                                           26     AAA GGC CTC TAA GAC AAG CT                                                                        "                                                           2285                  2266                                           27     GCC AGC ATG TGC ACC GTG AA                                                                        "                                                           2250                  2231                                           28     ACA CCC CAG GCT CAA CGA TG                                                                        "                                                           2186                  2167                                           29     CCG AAG CTT ACT CAC AAT TT                                                                        "                                                           2569                  2550                                           ______________________________________                                    

The following antisense oligonucleotides are targeted to the3'-untranslated region of PKC-β Type II:

                  TABLE 4                                                         ______________________________________                                        OLIGONUCLEOTIDES TARGETED TO PKC-β TYPE II                               SEQ. ID                                                                              Sequence            Target                                             ______________________________________                                        30     ACT TAG CTC TTG ACT TCG GG                                                                        3' Untranslated                                             2160                  2141                                           31     ATG CTG CGG AAA ATA AAT TG                                                                        "                                                           2420                  2401                                           32     AAT TTA TTT TGA GCA TGT TC                                                                        "                                                           2663                  2644                                           33     TTT GGG GAT GAG GGT GAG CA                                                                        "                                                           2843                  2824                                           34     CCC ATT CCC ACA GGC CTG AG                                                                        "                                                           3137                  3118                                           ______________________________________                                    

PKC-γ:

Sequence data are from Coussens et al., Science 233:859-866 (1986);Genbank accession number M13977. Sequences are numbered from the first5' base sequenced in the CDNA. The full sequence is not available: theextreme 3' end of the open reading frame and the 3' untranslated regionare missing. Consequently these regions are not presently available asantisense targets.

                  TABLE 5                                                         ______________________________________                                        OLIGONUCLEOTIDES TARGETED TO PKC-γ                                      SEQ. ID                                                                              Sequence            Target                                             ______________________________________                                        35     CGG AGC GCG CCA GGC AGG GA                                                                        5' Untranslated                                             51                      32                                           36     CCT TTT CCC AGA CCA GCC AT                                                                        Translation init.                                           215                    196                                           37     GGC CCC AGA AAC GTA GCA GG                                                                        5' of start codon                                           195                    176                                           38     GGA TCC TGC CTT TCT TGG GG                                                                        5' Untranslated                                             170                    151                                           39     CAG CCA TGG CCC CAG AAA CG                                                                        Translation init.                                           202                    183                                           ______________________________________                                    

PKC-η:

Sequence data for PKC-η are from Bacher and colleagues [Bacher et al.,Mol. Cell. Biol. 11:126-133 (1991)]; Genbank accession number M55284.They assign their isozyme the name PKC-L; however the sequence is almostidentical to that of mouse PKC-η, so the latter nomenclature is usedhere for consistency. Sequences are numbered from the first 5' basesequenced in the cDNA.

                  TABLE 6                                                         ______________________________________                                        OLIGONUCLEOTIDES TARGETED TO PKC-η                                        SEQ.                                                                          ID    Sequence           Target                                               ______________________________________                                        40    CGA CAT GCC GGC GCC GCT GC                                                                       Translation init.                                            172                    153                                            41    CAG ACG ACA TGC CGG CGC CG                                                                       "                                                            176                    157                                            42    GCC TGC TTC GCA GCG GGA GA                                                                       "                                                            138                    119                                            43    ACA GGT GCA GGA GTC GAG GC                                                                       "                                                            86                      67                                            44    GTC CCG TCT CAG GCC AGC CC                                                                       "                                                            111                     92                                            45    CCT CAC CGA TGC GGA CCC TC                                                                       "                                                            221                    202                                            46    ATT GAA CTT CAT GGT GCC AG                                                                       "                                                            193                    174                                            47    TCT CAC TCC CCA TAA GGC TA                                                                       3' Untranslated                                              2046                  2027                                            48    TTC CTT TGG GTT CTC GTG CC                                                                       "                                                            2067                  2048                                            49    TTC CAT CCT TCG ACA GAG TT                                                                       "                                                            2353                  2336                                            50    AGG CTG ATG CTG GGA AGG TC                                                                       "                                                            2300                  2281                                            51    GTT CTA AGG CTG ATG CTG GG                                                                       "                                                            2306                  2287                                            ______________________________________                                    

Example 5 Dose Response of Phosphorothioate/2'--O--MethylOligonucleotide Effects on PKC-α Protein Synthesis

A series of phosphorothioate, fully 2'--O--methyl oligonucleotideshaving SEQ ID NO: 1, 2, 3 and 5 were synthesized. A549 cells weretreated with 500 nM PDBu for 18 hours to downregulate PKC-α synthesis,washed to remove PDBu and then treated with oligonucleotide andDOTMA/DOPE cationic liposomes. Medium was replaced after four hours andthe cells were allowed to recover for another 20 hours. Proteins wereextracted and PKC-α protein levels were determined by immunoblotting asdescribed in Example 3. Results were quantified with a phosphorimager(Molecular Dynamics, Sunnyvale Calif.) and are shown in FIG. 2 expressedas percent of control (saline treatment). ISIS 4649 (SEQ ID NO: 3;squares) reduced PKC-α protein levels by 85-90% at 500 nM and had anIC50 of approximately 260 nM.

Example 6 Effect of Antisense Oligonucleotides on PKC-α mRNA Levels

A549 cells were treated with phosphorothioate oligonucleotides at 500 nMfor four hours in the presence of the cationic lipids DOTMA/DOPE, washedand allowed to recover for an additional 20 hours. Total RNA wasextracted and 20 μg of each was resolved on 1.2% gels and transferred tonylon membranes. These blots were probed with a ³² P radiolabeled PKC-αcDNA probe and then stripped and reprobed with a radiolabeled G3PDHprobe to confirm equal RNA loading. Each oligonucleotide (3520, 3521,3522 and 3527) was used in duplicate. The two major PKC-α transcripts(8.5 kb and 4.0 kb) were examined and quantified with a PhosphorImager(Molecular Dynamics, Sunnyvale Calif.). Results are shown in FIG. 3.Oligonucleotides 3521 (SEQ ID NO: 2), 3522 (SEQ ID NO: 3) and 3527 (SEQID NO: 5) gave better than 50% reduction of PKC-α mRNA levels.Oligonucleotides 3521 and 3527 gave approximately 80% reduction of thesmaller transcript and over 90% reduction of the larger transcript.

Example 7 Chimeric (Deoxy Gapped) 2'--O--Methyl Oligonucleotides

Oligonucleotides 3521 (SEQ ID NO: 2), 3522 (SEQ ID NO: 3) and 3527 (SEQID NO: 5) were chosen for further study and modification.Oligonucleotides having these sequences were synthesized as uniformlyphosphorothioate chimeric oligonucleotides having a centered deoxy gapof various lengths flanked by 2'--O--methylated regions. Theseoligonucleotides (500 nM concentration) were tested for effects on PKC-αmRNA levels by Northern blot analysis. Results are shown in FIG. 4.Deoxy gaps of eight nucleotides or more gave maximal reduction of PKC-αmRNA levels (both transcripts) in all cases. The oligo-nucleotide havingSEQ ID NO: 3 reduced PKC-α mRNA by approximately 83% with a deoxy gaplength of four nucleotides, and gave nearly complete reduction of PKC-αmRNA with a deoxy gap length of six or more.

Dose-response curves for these oligonucleotides are shown in FIG. 5. The2'--O--methyl chimeric oligonucleotides with four- or six-nucleotidedeoxy gaps have an IC50 for PKC-α mRNA reduction (concentration ofoligonucleotide needed to give a 50% reduction in PKC-α mRNA levels) of200-250 nM, as did the full-deoxy oligonucleotide (all arephosphorothioates throughout). The 2'--O--methyl chimericoligonucleotide with an 8-nucleotide deoxy gap had an IC50 ofapproximately 85 nM.

Several variations of this chimeric oligonucleotide (SEQ. ID NO: 3) werecompared for ability to lower PKC-α mRNA levels. These oligonucleotidesare shown in Table 7.

                  TABLE 7                                                         ______________________________________                                        Chimeric 2'-O-methyl/deoxy P=S oligonucleotides                               bold = 2'-O-methyl; s = P=S linkage,                                          o = P=O linkage                                                                                                  SEQ                                                                             ID                                       OLIGO #                                                                              SEQUENCE                    NO:                                        ______________________________________                                        3522   AsAsAsAsCsGsTsCsAsGsCsCsAsTsGsGsTsCsCsC                                                                   3                                          5352   AsAsAsAsCsGsTsCsAsGsCsCsAsTsGsGsTsCsCsC                                                                   3                                          6996   AoAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCoC                                                                   3                                          7008   AsAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCsC                                                                   3                                          7024   AsAoAoAoCoGsToCsAoGsCoCsAsTsGoGoToCoCsC                                                                   3                                          ______________________________________                                    

Effects of these oligonucleotides on PKC-α mRNA levels is shown in FIG.6. Oligonucleotides 7008, 3522 and 5352 show reduction of PKC-α mRNA,with 5352 being most active.

A series of 2'--O--propyl chimeric oligonucleotides was synthesizedhaving SEQ ID NO: 3. These oligonucleotides are shown in Table 8.

                  TABLE 8                                                         ______________________________________                                        Chimeric 2'-O-propyl/deoxy P=S oligonucleotides                               bold = 2+-O-propyl; s = P=S linkage,                                          o = P=O linkage                                                                                                  SEQ                                                                                  ID                                  OLIGO #                                                                              SEQUENCE                    NO:                                        ______________________________________                                        7199   AsAsAsAsCsGsTsCsAsGsCsCsAsTsGsGsTsCsCsC                                                                   3                                          7273   AoAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCoC                                                                   3                                          7294   AsAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCsC                                                                   3                                          7295   AsAoAoAoCoGsToCsAoGsCoCsAsTsGoGoToCoCsC                                                                   3                                          ______________________________________                                    

These 2'--O--propyl chimeric oligonucleotides were compared to the2'--O--methyl chimeric oligonucleotides. Oligonucleotides 7273 and 7294were more active than their 2'--O--methyl counterparts at lowering PKC-αmRNA levels. This is shown in FIGS. 7 and 8.

Example 8 Additional Oligonucleotides which Decrease PKC-α mRNA:

Additional phosphorothioate oligonucleotides targeted to the human PKC-α3' untranslated region were designed and synthesized. These sequencesare shown in Table 9.

                                      TABLE 9                                     __________________________________________________________________________    Chimeric 2'-O-propyl/deoxy P=S oligonucleotides                               targeted to PKC-α 3'-UTR                                                bold = 2'-O-propyl; s = P=S linkage, o = P=O linkage                          OLIGO #                                                                            SEQUENCE                 SEQ ID NO:                                      __________________________________________________________________________    6632 TsTsCs TsCsGs CsTsGs GsTsGs AsGsTs TsTsC 52                              6653 TsTsCs TsCsGs CsTsGs GsTsGs AsGsTs TsTsC 52                              6665 ToToCo TsCsGs CsTsGs GsTsGs AsGsTo ToToC 52                              7082 TsCsTs CsGsCs TsGsGs TsGsAs GsTsTs TsC 53                                7083 TsCsTs CsGsCs TsGsGs TsGsAs GsTsTs TsC 53                                7084 ToCoTo CsGsCs TsGsGs TsGsAs GsToTo ToC 53                                __________________________________________________________________________

As shown in FIG. 9, oligonucleotides 6632, 6653, 7082 and 7083 are mostactive in reducing PKC-α mRNA levels.

Example 9 Culture of Human A549 Lung Tumor Cells

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (Bethesda, Md.). Cells were grown in Dulbecco'sModified Eagle's Medium (Irvine Scientific, Irvine Calif.) containing 1gm glucose/liter and 10% fetal calf serum (Irvine Scientific). Cellswere trypsinized and washed and resuspended in the same medium forintroduction into mice.

Example 10 Effect of ISIS 3521 on the Growth of Human A549 Tumor Cellsin Nude Mice

200 μl of A549 cells (5×10⁶ cells) were implanted subcutaneously in theinner thigh of nude mice. ISIS 3521, a phosphorothioate oligonucleotidewith Sequence ID NO 2 was administered twice weekly for four weeks,beginning one week following tumor cell inoculation. Oligonucleotideswere formulated with cationic lipids (DMRIE/DOPE) and givensubcutaneously in the vicinity of the tumor. Oligonucleotide dosage was5 mg/kg with 60 mg/kg cationic lipid. Tumor size was recorded weekly.

As shown in FIG. 10, tumor growth was almost completely inhibited in twoof the three mice, and reduced compared to control in the third mouse.This inhibition of tumor growth by ISIS 3521 is statisticallysignificant. The control oligonucleotide (ISIS 1082) is a 21-merphosphorothioate oligonucleotide without significant sequence homologyto the PKC mRNA target.

Administration of oligonucleotides to mice whose tumors had alreadyreached detectable size had no discernable effect on subsequent tumorgrowth.

Example 11 Effect of Antisense Oligonucleotides on Growth of HumanMDA-MB231 Tumors in Nude Mice

MDA-MB231 human breast carcinoma cells were obtained from the AmericanType Culture Collection (Bethesda, Md.). Serially transplanted MDA-MB231tumors were established subcutaneously in nude mice. Beginning two weekslater, oligonucleotides 3521 and 3527, a phosphorothioateoligonucleotide having Sequence ID NO. 5, in saline, were administeredintravenously daily for 14 days at dosages of 60 mg/kg and 6 mg/kg.Control oligonucleotide ISIS 1082 was also administered at these doses,and a saline control was also given. Tumor growth rates wre monitoredfor the two-week period of oligonucleotide administration. As shown inFIG. 11, both PKC-α oligonucleotides (3521 and 3527) significantlyinhibit tumor growth at dosages of 60 mg/kg and 6 mg/kg. The controloligonucleotide (ISIS 1082) also showed some reduction in tumor growth,but this effect was less than with antisense oligonucleotides even athigh doses, and considerably less at the lower dose. A lower-dose studywas conducted using the same oligonucleotides at 6 mg/kg and 0.6 mg/kg.At 0.6 mg/kg ISIS 3521 significantly reduced tumor growth. At thisconcentration, ISIS 3527 also reduced tumor growth, but this result wasnot statistically--significant.

Example 12 Effect of Oligonucleotides on the Growth of Murine Lewis LungCarcinoma in Mice

Serially transplanted murine Lewis lung carcinomas were established inmice. Oligonucleotides 3521 and 3527 were administered intravenouslyevery day for 14 days at doses of 6 mg/kg and 0.6 mg/kg. Tumor growthrates were monitored for the two-week period of oligonucleotideadministration. As expected, these oligonucleotides, which are targetedto human PKC sequences, had insignificant effects on the mouse-derivedtumors.

Example 13 Effects of Antisense Oligonucleotide ISIS 4189 on EndogenousPKC-α Expression in Hairless Mice

In order to study oligonucleotide effects on endogenous PKC mRNA levelsin normal animals, it was necessary to employ an oligonucleotidecomplementary to the murine PKC-α. ISIS 4189 is a 20-merphosphorothioate oligonucleotide targeted to the AUG codon of mousePKC-α. This region is without homology to the human PKC sequence and theoligonucleotide has no effect on expression of PKC-α in human cells.ISIS 4189 has an IC50 of 200 nM for mRNA reduction in C127 mouse breastepithelial cells. ISIS 4189 in saline was administered intraperitoneallyto hairless mice at concentrations of 1, 10 or 100 mg/kg body weight.Injections were given daily for seven days. Tissues from liver, kidney,spleen, lung and skin were removed and PKC-α mRNA and protein levelswere determined. Histopathological examination was also performed onliver, kidney and lung samples. ISIS 4189 at 100 mg/kg inhibitedendogenous PKC-α mRNA levels in the mouse liver to 10-15% of control(saline) levels.

Example 14 Screening of Antisense Oligonucleotides Complementary toHuman PKC-η

A series of 20-mer phosphorothioate oligonucleotides complementary tohuman PKC-η were synthesized. These oligonucleotides were screened at aconcentration of 500 nM for ability to decrease PKC-η mRNA levels inhuman A549 cells, using a Northern blot assay. The oligonucleotidesequences are shown in Table 10 and the results are shown in FIG. 12.

                  TABLE 10                                                        ______________________________________                                        OLIGONUCLEOTIDES TARGETED TO HUMAN PKC-η mRNA                                                                 SEQ ID                                    ISIS#  Sequence             Target  NO:                                       ______________________________________                                        6431   CGA CAT GCC GGC GCC GCT GC                                                                         AUG     40                                        6442   CAG ACG ACA TGC CGG CGC CG                                                                         AUG     41                                        6443   GCC TGC TTC GCA GCG GGA GA                                                                         5' UTR  42                                        6432   ACA GGT GCA GGA GTC GAG GC                                                                         5' UTR  43                                        6433   GTC CCG TCT CAG GCC AGC CC5'                                                                       UTR     44                                        6435   CCT CAC CGA TGC GGA CCC TC                                                                         Coding  45                                        6441   ATT GAA CTT CAT GGT GCC AG                                                                         Coding  46                                        6581   TCT CAC TCC CCA TAA GGC TA                                                                         3' UTR  47                                        6580   TTC CTT TGG GTT CTC GTG CC                                                                         3' UTR  48                                        6436   AAC TCG AGG TGG CCG CCG TC                                                                         Coding  54                                        6434   CGC CTT CGC ATA GCC CTT TG                                                                         Coding  55                                        6444   GGA AGG GGT GAT TGC GGG CC                                                                         Coding  56                                        6445   AAC ACG CCC ATT GCC CAC CA                                                                         Coding  57                                        6446   GTC TCA AGA TGG CGT GCT CG                                                                         Coding  58                                        6553   GCG ATG GTT CAG CTG GGC GG                                                                         Coding  59                                        6605   GCC CTC TCT CTC ACT CCC CA                                                                         3' UTR  60                                        6579   CTG GGA AGG TCC GAT AGA GG                                                                         3' UTR  61                                        6603   AAG GCT GAT GCT GGG AAG GT                                                                         3' UTR  62                                        ______________________________________                                    

Oligonucleotides 6432, 6443, 6431, 6442, 6435, 6434, 6445, 6553, 6581and 6603 reduced PKC-η mRNA levels by greater than 50%. The most potentoligonucleotides were ISIS 6581 (targeting 3' untranslated region) andISIS 6445 (targeting coding region) which gave nearly complete loss ofPKC mRNA in this assay.

Example 15 Screening of Antisense Oligonucleotides Complementary toHuman PKC-ζ

A series of 20-mer phosphorothioate oligonucleotides complementary tohuman PKC-ζ were synthesized as described in Example 1. The source ofthe target sequence was Genbank locus HSPKCZ, accession number Z15108(Hug, H.). These oligonucleotides were screened at a concentration of500 nM for ability to decrease PKC-ζ mRNA levels in human A549 cells,substantially as described in Example 6 using a Northern blot assay. Theoligonucleotide sequences and results of the screen are shown in Table11.

                  TABLE 11                                                        ______________________________________                                        INHIBITION OF mRNA EXPRESSION IN HUMAN A549 CELLS                             USING ANTISENSE OLIGONUCLEOTIDES COMPLEMENTARY                                TO PKC-Z                                                                                              Target         Seq.                                   Oligo #                                                                              Sequence         region  % Inhib.                                                                             ID                                     ______________________________________                                        9007   CGCCGCTCCCTTCCATCTTG                                                                           AUG     70     63                                     9008   CCCCGTAATGCGCCTTGAGG                                                                           Coding  68     64                                     9009   CTGTCCACCCACTTGAGGGT                                                                           Coding  19     65                                     9010   GCTTCCTCCATCTTCTGGCT                                                                           Coding  35     66                                     9011   CGGTACAGCTTCCTCCATCT                                                                           Coding  58     67                                     9012   TTGGAAGAGGTGGCCGTTGG                                                                           Coding  80     68                                     9013   CCTGTTAAAGCGCTTGGCTT                                                                           Coding  71     69                                     9014   TGCAGGTCAGCGGGACGAGG                                                                           Coding  41     70                                     9015   GCTCTTGGGAAGGCATGACA                                                                           Coding  59     71                                     9016   TTCTTCAACCGCACCAGGAG                                                                           Coding  0      72                                     9017   TTCTTCAACCGCACCAGGAG                                                                           Coding  73     73                                     9018   CTCTGCCTCTGCATGTGGAA                                                                           Coding  63     74                                     9019   TCCTTGCACATGCCGTAGTC                                                                           Coding  31     75                                     9020   TCCACGCTGAACCCGTACTC                                                                           Coding  80     76                                     9021   GGAGCGCCCGGCCATCATCT                                                                           Coding  81     77                                     9022   GGGCTCGCTGGTGAACTGTG                                                                           Coding  83     78                                     9023   GACGCACGCGGCCTCACACC                                                                           Stop    82     79                                     9024   GGGTCAATCACGCGTGTCCA                                                                           3' UTR  70     80                                     9025   TCGGAGCCGTGCCCAGCCTG                                                                           3' UTR  82     81                                     9026   CGGGCCAGGTGTGAGGGACT                                                                           3' UTR  40     82                                     9027   CCGCGACGCAGGCACAGCAG                                                                           3' UTR  38     83                                     9028   TGGAAACCGCATGACAGCCC                                                                           3' UTR  54     84                                     9029   GGTCAGTGCATCGAGTTCTG                                                                           3' UTR  79     85                                     ______________________________________                                    

In this experiment, oligonucleotides 9007, 9008, 9011, 9012, 9013, 9015,9017, 9018, 9020, 9021, 9022, 9023, 9024, 9025, 9028 and 9029 showed atleast 50% inhibition of mRNA levels and are presently preferred.

Example 16 Screening of Antisense Oligonucleotides Complementary toHuman PKC-ε

A series of 20-mer phosphorothioate oligonucleotides complementary tohuman PKC-ε were synthesized as described in Example 1. The source ofthe target sequence was Genbank locus HSPKCE, accession number X65293(Burns et al.). These oligonucleotides were screened at a concentrationof 500 nM for ability to decrease PKC-ε mRNA levels in human A549 cells,substantially as described in Example 6 using a Northern blot assay. Theoligonucleotide sequences and results of the screen are shown in Table12.

                  TABLE 12                                                        ______________________________________                                        INHIBITION OF mRNA EXPRESSION IN HUMAN A549 CELLS                             USING ANTISENSE OLIGONUCLEOTIDES COMPLEMENTARY                                TO PKC-E MRNA                                                                                         Target         Seq.                                   Oligo #                                                                              Sequence         region  % Inhib                                                                              ID                                     ______________________________________                                        7933   ACTACCATGGTCGGGGCGGG                                                                           AUG     0      86                                     7934   GTCCCACCGCATGGCGCAGC                                                                           Coding  0      87                                     7935   GTTTGGCCGATGCGCGAGTC                                                                           Coding  0      88                                     7936   TGCAGTTGGCCACGAAGTCG                                                                           Coding  0      89                                     8032   GTGGGGCATGTTGACGCTGA                                                                           Coding  0      90                                     8031   CCAGAGCAGGGACCCACAGT                                                                           Coding  0      91                                     7939   TCTCCTCGGTTGTCAAATGA                                                                           Coding  0      92                                     7940   CGGTGCTCCTCTCCTCGGTT                                                                           Coding  0      93                                     7941   AGCCAAAATCCTCTTCTCTG                                                                           Coding  0      94                                     7942   CATGAGGGCCGATGTGACCT                                                                           Coding  62     95                                     7943   ATCCCTTCCTTGCACATCCC                                                                           Coding  4      96                                     7944   CCCCAGGGCCCACCAGTCCA                                                                           Coding  42     97                                     7945   AGCACCCCCAGGGCCCACCA                                                                           Coding  56     98                                     7946   CGTACATCAGCACCCCCAGG                                                                           Coding  55     99                                     7947   CCAGCCATCATCTCGTACAT                                                                           Coding  15     100                                    7948   TGCCACACAGCCCAGGCGCA                                                                           Coding  55     101                                    7949   TCAGGGCATCAGGTCTTCAC                                                                           Stop    0      102                                    7950   CTCTCAGGGCATCAGGTCTT                                                                           Stop    0      103                                    ______________________________________                                    

In this experiment, oligonucleotides 7942, 7944, 7945, 7946 and 7948showed at least 40% inhibition of mRNA levels and are presentlypreferred.

Example 17 DNA Sequencing of the 3' Untranslated Region of Human PKCα

A549 cells (obtained from the American Type Culture Collection, BethesdaMd.) were grown to confluence in 6-well plates (Falcon Labware, LincolnPark, N.J.) in Dulbecco's modified Eagle's medium (DME) containing 1 gglucose/liter and 10% fetal calf serum (FCS, Irvine Scientific, SantaAna, Calif.). Cells were harvested and total RNA was isolated usingstandard methods. Sambrook, J., Fritsch, E., and T. Maniatis (1989).Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., Ch. 7).

cDNA was made from the RNA using the 3' RACE technique of Frohman et al.[Frohman, M. A., Dush, M. K. and G. R. Martin (1988) Proc. Natl. Acad.Sci. U.S.A. 85:8998-9002] and the 3' RACE kit from Gibco/BRL (Bethesda,Md.). For making the first strand of cDNA, an oligo dT primer was used.For subsequent amplification from the site of the poly(A) tail, theoligonucleotide provided in the kit or an identical oligonucleotide(ISIS 5586; SEQ ID NO: 107:5'-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3'). For amplification from theinterior of the known sequence, ISIS 6288 was used (SEQ ID NO: 108:5'-GGGGTAGAATGCGGCGGCAGTATGAAACTCACCAGCG-3'). The DNA resulting from thePCR reaction was gel-purified, digested with Sal I and Bcl I, and thencloned into the Bluescript plasmid (Stratagene, La Jolla, Calif.) usingstandard techniques (Sambrook et al., 1989). The cloned DNA wassequenced using a Sequenase Kit from USB.

The new sequence obtained, from the Bcl I site near the 3' end of thepreviously known sequence (GenBank accession number x52479) to the mostfrequently obtained site of polyadenylation is shown as nucleotides56-1136 in FIG. 13. This site is believed to be the 3' end of the short(4 kb) PKCα message.

To extend this sequence and hence obtain sequences specific for the longPKCα message (8.5 kb), the technique of Inverse PCR was performed.Ochman, H., Gerber, A. S. and D. L. Hartl (1988) Genetics 120:621-623.This technique was performed three times using a three sets of primersand restriction enzymes. Each round resulted in about 200 bases of newsequence; the total of the new sequence (SEQ ID NO: 104) is shown inbold type (nucleotides 1137-1812) in FIG. 13. This sequence is shownextending in the 3' direction beginning at the Bcl I site (TGATCA) nearthe end of the previously published PKCα cDNA sequence. Finkenzeller etal., Nucl. Acids Res. 18:2183 (1990); Genbank accession number X52479.Newly determined sequences begin at nucleotide 56 and are underlined(SEQ ID NO:105). The most common site of polyadenylation, believed to bethe 3' end of the short (4 kb) mRNA transcript, is at nucleotide 1136.Sequences downstream from this site, and therefore unique to the longmessage, are in bold (SEQ ID NO:106).

Example 18 Antisense Oligonucleotides Targeted to Novel Sequences in the3' UTR of PKCα

A series of phosphorothioate antisense oligonucleotides, complementaryto the novel sequence obtained as described in Example 17, were designedand synthesized. These oligonucleotides were screened on the basis oftheir ability to cause the reduction or elimination of PKCα RNA in A549cells 24 hours after the start of treatment. A549 cells were treatedwith phosphorothioate oligonucleotides at 500 nM for four hours in thepresence of the cationic lipids DOTMA/DOPE, washed and allowed torecover for an additional 20 hours. Total RNA was extracted and 20 μg ofeach was resolved on 1.2% gels and transferred to nylon membranes. Theseblots were probed with a ³² p radiolabeled PKC-α cDNA probe and thenstripped and reprobed with a radiolabeled G3PDH probe to confirm equalRNA loading. The two major PKC-α transcripts (8.5 kb and 4.0 kb) wereexamined and quantified with a PhosphorImager (Molecular Dynamics,Sunnyvale Calif.). The oligonucleotides and their activities are shownin Table 13.

                  TABLE 13                                                        ______________________________________                                        Inhibition of PKCα mRNA (both long and short) by                        phosphorothioate antisense oligonucleotides (500                              nM) Expressed as percent of control mRNA level                                                                Target                                                                              SEQ                                     ISIS# Sequence         Activity region                                                                              ID NO:                                  ______________________________________                                        7416  CAGTGCCCATGTGCAGGGAG                                                                           100%     PKCα long mRNA                                                                109                                     7417  AGAACCTGCACAAATAGAGC                                                                           100%     PKCα long mRNA                                                                110                                     7418  AGAAACAAGAACCTGCACAA                                                                           100%     PKCα long mRNA                                                                111                                     7419  GCAAGGGATTCAGCTAAAAC                                                                           100%     PKCα long mRNA                                                                112                                     7420  AGGGAGGGAAAGCACAGAAG                                                                           100%     PKCα long mRNA                                                                113                                     7902  AGGGAGGGAAAGCACAGAAG                                                                            90%     PKCα long mRNA                                                                113                                     7907  TCAGCTCAAAAATAGTCCTC                                                                            85%     PKCα long mRNA                                                                114                                     7908  CGAAAGGTGACATGAAGAAA                                                                           100%     PKCα long mRNA                                                                115                                     7909  GGCGGAGGAACCAGGACGAA                                                                            90%     PKCα long mRNA                                                                116                                     7911  GCAATGCCACGTGTGTACCA                                                                            50%     PKCα long mRNA                                                                117                                     7912  TGCAAAACGTATTAAAATCC                                                                           100%     PKCα short mRNA                                                               118                                     7913  TTATAAACATGCAAAATTCA                                                                           100%     PKCα short mRNA                                                               119                                     ______________________________________                                    

ISIS 7911 (SEQ ID NO: 117) reduced PKCα mRNA levels (both long and shortmessages) in this preliminary experiment by 50% compared to control.This oligonucleotide is therefore preferred. Further analysisdemonstrated that ISIS 7911 selectively reduced the amount of long (8.5kb) message during the first six hours of treatment, with a fourfoldselectivity at 3 hours post-treatment. By 12 hours after treatment withISIS 7911, levels of both messages were reduced by over 80%. Time-coursedata are shown in FIG. 14.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 122                                           - (2) INFORMATION FOR SEQ ID NO: 1:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 1:                           # 20               CTCC                                                       - (2) INFORMATION FOR SEQ ID NO: 2:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 2:                           # 20               TTCA                                                       - (2) INFORMATION FOR SEQ ID NO: 3:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 3:                           # 20               TCCC                                                       - (2) INFORMATION FOR SEQ ID NO: 4:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 4:                           # 20               CGGG                                                       - (2) INFORMATION FOR SEQ ID NO: 5:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 5:                           # 20               GATC                                                       - (2) INFORMATION FOR SEQ ID NO: 6:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 6:                           # 20               CCAT                                                       - (2) INFORMATION FOR SEQ ID NO: 7:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 7:                           # 20               TTGC                                                       - (2) INFORMATION FOR SEQ ID NO: 8:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 8:                           # 20               GGAG                                                       - (2) INFORMATION FOR SEQ ID NO: 9:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 9:                           # 20               TTGA                                                       - (2) INFORMATION FOR SEQ ID NO: 10:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 10:                          # 20               TGTT                                                       - (2) INFORMATION FOR SEQ ID NO: 11:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 11:                          # 20               GGGG                                                       - (2) INFORMATION FOR SEQ ID NO: 12:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 12:                          # 20               TTTT                                                       - (2) INFORMATION FOR SEQ ID NO: 13:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 13:                          # 20               ATGG                                                       - (2) INFORMATION FOR SEQ ID NO: 14:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 14:                          # 20               ACAG                                                       - (2) INFORMATION FOR SEQ ID NO: 15:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 15:                          # 20               GTAA                                                       - (2) INFORMATION FOR SEQ ID NO: 16:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 16:                          # 20               GGGG                                                       - (2) INFORMATION FOR SEQ ID NO: 17:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 17:                          # 20               CCCC                                                       - (2) INFORMATION FOR SEQ ID NO: 18:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 18:                          # 20               CCCG                                                       - (2) INFORMATION FOR SEQ ID NO: 19:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 19:                          # 20               CGGC                                                       - (2) INFORMATION FOR SEQ ID NO: 20:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 20:                          # 20               CCGT                                                       - (2) INFORMATION FOR SEQ ID NO: 21:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 21:                          # 20               AGCC                                                       - (2) INFORMATION FOR SEQ ID NO: 22:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 22:                          # 20               GAGC                                                       - (2) INFORMATION FOR SEQ ID NO: 23:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 23:                          # 20               CGGG                                                       - (2) INFORMATION FOR SEQ ID NO: 24:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 24:                          # 20               TCGG                                                       - (2) INFORMATION FOR SEQ ID NO: 25:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 25:                          # 20               CTAC                                                       - (2) INFORMATION FOR SEQ ID NO: 26:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 26:                          # 20               AGCT                                                       - (2) INFORMATION FOR SEQ ID NO: 27:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 27:                          # 20               TGAA                                                       - (2) INFORMATION FOR SEQ ID NO: 28:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 28:                          # 20               GATG                                                       - (2) INFORMATION FOR SEQ ID NO: 29:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 29:                          # 20               ATTT                                                       - (2) INFORMATION FOR SEQ ID NO: 30:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 30:                          # 20               CGGG                                                       - (2) INFORMATION FOR SEQ ID NO: 31:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 31:                          # 20               ATTG                                                       - (2) INFORMATION FOR SEQ ID NO: 32:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 32:                          # 20               GTTC                                                       - (2) INFORMATION FOR SEQ ID NO: 33:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 33:                          # 20               AGCA                                                       - (2) INFORMATION FOR SEQ ID NO: 34:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 34:                          # 20               TGAG                                                       - (2) INFORMATION FOR SEQ ID NO: 35:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 35:                          # 20               GGGA                                                       - (2) INFORMATION FOR SEQ ID NO: 36:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 36:                          # 20               CCAT                                                       - (2) INFORMATION FOR SEQ ID NO: 37:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 37:                          # 20               CAGG                                                       - (2) INFORMATION FOR SEQ ID NO: 38:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 38:                          # 20               GGGG                                                       - (2) INFORMATION FOR SEQ ID NO: 39:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 39:                          # 20               AACG                                                       - (2) INFORMATION FOR SEQ ID NO: 40:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 40:                          # 20               CTGC                                                       - (2) INFORMATION FOR SEQ ID NO: 41:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 41:                          # 20               GCCG                                                       - (2) INFORMATION FOR SEQ ID NO: 42:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 42:                          # 20               GAGA                                                       - (2) INFORMATION FOR SEQ ID NO: 43:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 43:                          # 20               AGGC                                                       - (2) INFORMATION FOR SEQ ID NO: 44:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 44:                          # 20               GCCC                                                       - (2) INFORMATION FOR SEQ ID NO: 45:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 45:                          # 20               CCTC                                                       - (2) INFORMATION FOR SEQ ID NO: 46:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 46:                          # 20               CCAG                                                       - (2) INFORMATION FOR SEQ ID NO: 47:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 47:                          # 20               GCTA                                                       - (2) INFORMATION FOR SEQ ID NO: 48:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 48:                          # 20               TGCC                                                       - (2) INFORMATION FOR SEQ ID NO: 49:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 49:                          # 20               AGTT                                                       - (2) INFORMATION FOR SEQ ID NO: 50:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 50:                          # 20               GGTC                                                       - (2) INFORMATION FOR SEQ ID NO: 51:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 51:                          # 20               TGGG                                                       - (2) INFORMATION FOR SEQ ID NO: 52:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #52:                           #  18              TC                                                         - (2) INFORMATION FOR SEQ ID NO: 53:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 17                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 53:                          #   17             C                                                          - (2) INFORMATION FOR SEQ ID NO: 54:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 54:                          # 20               CGTC                                                       - (2) INFORMATION FOR SEQ ID NO: 55:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 55:                          # 20               TTTG                                                       - (2) INFORMATION FOR SEQ ID NO: 56:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 56:                          # 20               GGCC                                                       - (2) INFORMATION FOR SEQ ID NO: 57:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 57:                          # 20               ACCA                                                       - (2) INFORMATION FOR SEQ ID NO: 58:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 58:                          # 20               CTCG                                                       - (2) INFORMATION FOR SEQ ID NO: 59:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 59:                          # 20               GCCC                                                       - (2) INFORMATION FOR SEQ ID NO: 60:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 60:                          # 20               CCCA                                                       - (2) INFORMATION FOR SEQ ID NO: 61:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 61:                          # 20               GAGG                                                       - (2) INFORMATION FOR SEQ ID NO: 62:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 62:                          # 20               AGGT                                                       - (2) INFORMATION FOR SEQ ID NO: 63 :                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 63:                          # 20               CTTG                                                       - (2) INFORMATION FOR SEQ ID NO:64 :                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 64:                          # 20               GAGG                                                       - (2) INFORMATION FOR SEQ ID NO: 65:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 65:                          # 20               GGGT                                                       - (2) INFORMATION FOR SEQ ID NO: 66:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 66:                          # 20               GGCT                                                       - (2) INFORMATION FOR SEQ ID NO: 67:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 67:                          # 20               ATCT                                                       - (2) INFORMATION FOR SEQ ID NO: 68:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 68:                          # 20               TTGG                                                       - (2) INFORMATION FOR SEQ ID NO: 69:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 69:                          # 20               GCTT                                                       - (2) INFORMATION FOR SEQ ID NO: 70:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 70:                          # 20               GAGG                                                       - (2) INFORMATION FOR SEQ ID NO: 71:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 71:                          # 20               GACA                                                       - (2) INFORMATION FOR SEQ ID NO: 72:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 72:                          # 20               GGAG                                                       - (2) INFORMATION FOR SEQ ID NO: 73:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 73:                          # 20               GGAG                                                       - (2) INFORMATION FOR SEQ ID NO: 74:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 74:                          # 20               GGAA                                                       - (2) INFORMATION FOR SEQ ID NO: 75:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 75:                          # 20               AGTC                                                       - (2) INFORMATION FOR SEQ ID NO: 76:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 76:                          # 20               ACTC                                                       - (2) INFORMATION FOR SEQ ID NO: 77:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 77:                          # 20               ATCT                                                       - (2) INFORMATION FOR SEQ ID NO: 78:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 78:                          # 20               TGTG                                                       - (2) INFORMATION FOR SEQ ID NO: 79:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 79:                          # 20               CACC                                                       - (2) INFORMATION FOR SEQ ID NO: 80:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 80:                          # 20               TCCA                                                       - (2) INFORMATION FOR SEQ ID NO: 81:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 81:                          # 20               CCTG                                                       - (2) INFORMATION FOR SEQ ID NO: 82:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 82:                          # 20               GACT                                                       - (2) INFORMATION FOR SEQ ID NO: 83:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 83:                          # 20               GCAG                                                       - (2) INFORMATION FOR SEQ ID NO: 84:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 84:                          # 20               GCCC                                                       - (2) INFORMATION FOR SEQ ID NO: 85:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 85:                          # 20               TCTG                                                       - (2) INFORMATION FOR SEQ ID NO: 86:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 86:                          # 20               CGGG                                                       - (2) INFORMATION FOR SEQ ID NO: 87:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 87:                          # 20               CAGC                                                       - (2) INFORMATION FOR SEQ ID NO: 88:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 88:                          # 20               AGTC                                                       - (2) INFORMATION FOR SEQ ID NO: 89:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 89:                          # 20               GTCG                                                       - (2) INFORMATION FOR SEQ ID NO: 90:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 90:                          # 20               CTGA                                                       - (2) INFORMATION FOR SEQ ID NO: 91:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 91:                          # 20               CAGT                                                       - (2) INFORMATION FOR SEQ ID NO: 92:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 92:                          # 20               ATGA                                                       - (2) INFORMATION FOR SEQ ID NO: 93:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 93:                          # 20               GGTT                                                       - (2) INFORMATION FOR SEQ ID NO: 94:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 94:                          # 20               TCTG                                                       - (2) INFORMATION FOR SEQ ID NO: 95:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 95:                          # 20               ACCT                                                       - (2) INFORMATION FOR SEQ ID NO: 96:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 96:                          # 20               TCCC                                                       - (2) INFORMATION FOR SEQ ID NO: 97:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 97:                          # 20               TCCA                                                       - (2) INFORMATION FOR SEQ ID NO: 98:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 98:                          # 20               ACCA                                                       - (2) INFORMATION FOR SEQ ID NO: 99:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 99:                          # 20               CAGG                                                       - (2) INFORMATION FOR SEQ ID NO: 100:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 100:                         # 20               ACAT                                                       - (2) INFORMATION FOR SEQ ID NO: 101:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 101:                         # 20               CGCA                                                       - (2) INFORMATION FOR SEQ ID NO: 102:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 102:                         # 20               TCAC                                                       - (2) INFORMATION FOR SEQ ID NO: 103:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 103:                         # 20               TCTT                                                       - (2) INFORMATION FOR SEQ ID NO:104:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1812 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:                               - TGATCAACTG TTCAGGGTCT CTCTCTTACA ACCAAGAACA TTATCTTAGT GG - #AAGATGGT         60                                                                          - ACGTCATGCT CAGTGTCCAG TTTAATTCTG TAGAAGTTAC GTCTGGCTCT AG - #GTTAACCC        120                                                                          - TTCCTAGAAA GCAAGCAGAC TGTTGCCCCA TTTTGGGTAC AATTTGATAT AC - #TTTCCATA        180                                                                          - CCCTCCATCT GTGGATTTTT CAGCATTGGA ATCCCCCAAC CAGAGATGTT AA - #AGTGAGCT        240                                                                          - GTCCCAGGAA ACATCTCCAC CCAAGACGTC TTTGGAATCC AAGAACAGGA AG - #CCAAGAGA        300                                                                          - GTGAGCAGGG AGGGATTGGG GGTGGGGGGA GGCCTCAAAA TACCGACTGC GT - #CCATTCTC        360                                                                          - TGCCTCCATG GAAACAGCCC CTAGAATCTG AAAGGCCGGG ATAAACCTAA TC - #ACTGTTCC        420                                                                          - CAAACATTGA CAAATCCTAA CCCAACCATG GTCCAGCAGT TACCAGTTTA AA - #CAAAAAAA        480                                                                          - ACCTCAGATG AGTGTTGGGT GAATCTGTCA TCTGGTACCC TCCTTGGTTG AT - #AACTGTCT        540                                                                          - TGATACTTTT CATTCTTTGT AAGAGGCCAA ATCGTCTAAG GACGTTGCTG AA - #CAAGCGTG        600                                                                          - TGAAATCATT TCAGATCAAG GATAAGCCAG TGTGTACATA TGTTCATTTT AA - #TCTCTGGG        660                                                                          - AGATTATTTT TCCATCCAGG GTGCCATCAG TAATCATGCC ACTACTCACC AG - #TGTTGTTC        720                                                                          - GCCAACACCC ACCCCCACAC ACACCAACAT TTTGCTGCCT ACCTTGTTAT CC - #TTCTCAAG        780                                                                          - AAGCTGAAGT GTACGCCCTC TCCCCTTTTG TGCTTATTTA TTTAATAGGC TG - #CAGTGTCG        840                                                                          - CTTATGAAAG TACGATGTAC AGTAACTTAA TGGAAGTGCT GACTCTAGCA TC - #AGCCTCTA        900                                                                          - CCGATTGATT TTCCTCCCTT CTCTAGCCCT GGATGTCCAC TTAGGGATAA AA - #AGAATATG        960                                                                          - GTTTTGGTTC CCATTTCTAG TTCACGTTGA ATGACAGGCC TGGAGCTGTA GA - #ATCAGGA        1020                                                                          - ACCCGGATGC CTAACAGCTC AAAGATGTTT TGTTAATAGA AGGATTTTAA TA - #CGTTTTG        1080                                                                          - AAATGCATCA TGCAATGAAT TTTGCATGTT TATAATAAAC CTTAATAACA AG - #TGAATAG        1140                                                                          - AGGATTTTAA TACGTTTTGC AAATGCATCA TGCAATGAAT TTTGCATGTT TA - #TAATAAA        1200                                                                          - CTTAATAACA AGTGAATCTA TATTATTGAT ATAATCGTAT CAAGTATAAA GA - #GAGTATT        1260                                                                          - TAATAATTTT ATAAGACACA ATTGTGCTCT ATTTGTGCAG GTTCTTGTTT CT - #AATCCTC        1320                                                                          - TTTCTAATTA AGTTTTAGCT GAATCCCTTG CTTCTGTGCT TTCCCTCCCT GC - #ACATGGG        1380                                                                          - ACTGTATCAG ATAGATTACT TTTTAAATGT AGATAAAATT TCAAAAATGA AT - #GGCTAGT        1440                                                                          - TACGTGATAG ATTAGGCTCT TACTACATAT GTGTGTGTAT ATATATGTAT TT - #GATTCTA        1500                                                                          - CTGCAAACAA ATTTTTATTG GTGAGGACTA TTTTTGAGCT GACACTCCCT CT - #TAGTTTC        1560                                                                          - TCATGTCACC TTTCGTCCTG GTTCCTCCGC CACTCTTCCT CTTGGGGACA AC - #AGGAAGT        1620                                                                          - TCTGATTCCA GTCTGGCCTA GTACGTTGGT ACACACGTGG CATTGCGCAG CA - #CCTGGGC        1680                                                                          - GACCTTTGTG TGTAGCGTGT GTGTGTGTTT CCTTCTTCCC TTCAGCCTGT GA - #CTGTTGC        1740                                                                          - GACTCCAGGG GTGGGAGGGA TGGGGAGACT CCCCTCTTGC TGTGTGTACT GG - #ACACGCA        1800                                                                          #     1812                                                                    - (2) INFORMATION FOR SEQ ID NO:105:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1757 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:                               - ATGGTACGTC ATGCTCAGTG TCCAGTTTAA TTCTGTAGAA GTTACGTCTG GC - #TCTAGGTT         60                                                                          - AACCCTTCCT AGAAAGCAAG CAGACTGTTG CCCCATTTTG GGTACAATTT GA - #TATACTTT        120                                                                          - CCATACCCTC CATCTGTGGA TTTTTCAGCA TTGGAATCCC CCAACCAGAG AT - #GTTAAAGT        180                                                                          - GAGCTGTCCC AGGAAACATC TCCACCCAAG ACGTCTTTGG AATCCAAGAA CA - #GGAAGCCA        240                                                                          - AGAGAGTGAG CAGGGAGGGA TTGGGGGTGG GGGGAGGCCT CAAAATACCG AC - #TGCGTCCA        300                                                                          - TTCTCTGCCT CCATGGAAAC AGCCCCTAGA ATCTGAAAGG CCGGGATAAA CC - #TAATCACT        360                                                                          - GTTCCCAAAC ATTGACAAAT CCTAACCCAA CCATGGTCCA GCAGTTACCA GT - #TTAAACAA        420                                                                          - AAAAAACCTC AGATGAGTGT TGGGTGAATC TGTCATCTGG TACCCTCCTT GG - #TTGATAAC        480                                                                          - TGTCTTGATA CTTTTCATTC TTTGTAAGAG GCCAAATCGT CTAAGGACGT TG - #CTGAACAA        540                                                                          - GCGTGTGAAA TCATTTCAGA TCAAGGATAA GCCAGTGTGT ACATATGTTC AT - #TTTAATCT        600                                                                          - CTGGGAGATT ATTTTTCCAT CCAGGGTGCC ATCAGTAATC ATGCCACTAC TC - #ACCAGTGT        660                                                                          - TGTTCGCCAA CACCCACCCC CACACACACC AACATTTTGC TGCCTACCTT GT - #TATCCTTC        720                                                                          - TCAAGAAGCT GAAGTGTACG CCCTCTCCCC TTTTGTGCTT ATTTATTTAA TA - #GGCTGCAG        780                                                                          - TGTCGCTTAT GAAAGTACGA TGTACAGTAA CTTAATGGAA GTGCTGACTC TA - #GCATCAGC        840                                                                          - CTCTACCGAT TGATTTTCCT CCCTTCTCTA GCCCTGGATG TCCACTTAGG GA - #TAAAAAGA        900                                                                          - ATATGGTTTT GGTTCCCATT TCTAGTTCAC GTTGAATGAC AGGCCTGGAG CT - #GTAGAATC        960                                                                          - AGGAAACCCG GATGCCTAAC AGCTCAAAGA TGTTTTGTTA ATAGAAGGAT TT - #TAATACG        1020                                                                          - TTTGCAAATG CATCATGCAA TGAATTTTGC ATGTTTATAA TAAACCTTAA TA - #ACAAGTG        1080                                                                          - ATAGAAGGAT TTTAATACGT TTTGCAAATG CATCATGCAA TGAATTTTGC AT - #GTTTATA        1140                                                                          - TAAACCTTAA TAACAAGTGA ATCTATATTA TTGATATAAT CGTATCAAGT AT - #AAAGAGA        1200                                                                          - TATTATAATA ATTTTATAAG ACACAATTGT GCTCTATTTG TGCAGGTTCT TG - #TTTCTAA        1260                                                                          - CCTCTTTTCT AATTAAGTTT TAGCTGAATC CCTTGCTTCT GTGCTTTCCC TC - #CCTGCAC        1320                                                                          - TGGGCACTGT ATCAGATAGA TTACTTTTTA AATGTAGATA AAATTTCAAA AA - #TGAATGG        1380                                                                          - TAGTTTACGT GATAGATTAG GCTCTTACTA CATATGTGTG TGTATATATA TG - #TATTTGA        1440                                                                          - TCTACCTGCA AACAAATTTT TATTGGTGAG GACTATTTTT GAGCTGACAC TC - #CCTCTTA        1500                                                                          - TTTCTTCATG TCACCTTTCG TCCTGGTTCC TCCGCCACTC TTCCTCTTGG GG - #ACAACAG        1560                                                                          - AAGTGTCTGA TTCCAGTCTG GCCTAGTACG TTGGTACACA CGTGGCATTG CG - #CAGCACC        1620                                                                          - GGGCTGACCT TTGTGTGTAG CGTGTGTGTG TGTTTCCTTC TTCCCTTCAG CC - #TGTGACT        1680                                                                          - TTGCTGACTC CAGGGGTGGG AGGGATGGGG AGACTCCCCT CTTGCTGTGT GT - #ACTGGAC        1740                                                                          # 1757             A                                                          - (2) INFORMATION FOR SEQ ID NO:106:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 676 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:                               - TAGAAGGATT TTAATACGTT TTGCAAATGC ATCATGCAAT GAATTTTGCA TG - #TTTATAAT         60                                                                          - AAACCTTAAT AACAAGTGAA TCTATATTAT TGATATAATC GTATCAAGTA TA - #AAGAGAGT        120                                                                          - ATTATAATAA TTTTATAAGA CACAATTGTG CTCTATTTGT GCAGGTTCTT GT - #TTCTAATC        180                                                                          - CTCTTTTCTA ATTAAGTTTT AGCTGAATCC CTTGCTTCTG TGCTTTCCCT CC - #CTGCACAT        240                                                                          - GGGCACTGTA TCAGATAGAT TACTTTTTAA ATGTAGATAA AATTTCAAAA AT - #GAATGGCT        300                                                                          - AGTTTACGTG ATAGATTAGG CTCTTACTAC ATATGTGTGT GTATATATAT GT - #ATTTGATT        360                                                                          - CTACCTGCAA ACAAATTTTT ATTGGTGAGG ACTATTTTTG AGCTGACACT CC - #CTCTTAGT        420                                                                          - TTCTTCATGT CACCTTTCGT CCTGGTTCCT CCGCCACTCT TCCTCTTGGG GA - #CAACAGGA        480                                                                          - AGTGTCTGAT TCCAGTCTGG CCTAGTACGT TGGTACACAC GTGGCATTGC GC - #AGCACCTG        540                                                                          - GGCTGACCTT TGTGTGTAGC GTGTGTGTGT GTTTCCTTCT TCCCTTCAGC CT - #GTGACTGT        600                                                                          - TGCTGACTCC AGGGGTGGGA GGGATGGGGA GACTCCCCTC TTGCTGTGTG TA - #CTGGACAC        660                                                                          #   676                                                                       - (2) INFORMATION FOR SEQ ID NO: 107:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 37                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: no                                                     -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #107:                          #      37          GTAC TTTTTTTTTT TTTTTTT                                    - (2) INFORMATION FOR SEQ ID NO: 108:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 37                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: no                                                     -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 108:                         #      37          GCAG TATGAAACTC ACCAGCG                                    - (2) INFORMATION FOR SEQ ID NO: 109:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 109:                         # 20               GGAG                                                       - (2) INFORMATION FOR SEQ ID NO: 110:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 110:                         # 20               GAGC                                                       - (2) INFORMATION FOR SEQ ID NO: 111:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 111:                         # 20               ACAA                                                       - (2) INFORMATION FOR SEQ ID NO: 112:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 112:                         # 20               AAAC                                                       - (2) INFORMATION FOR SEQ ID NO: 113:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 113:                         # 20               GAAG                                                       - (2) INFORMATION FOR SEQ ID NO: 114:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 114:                         # 20               CCTC                                                       - (2) INFORMATION FOR SEQ ID NO: 115:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 115:                         # 20               GAAA                                                       - (2) INFORMATION FOR SEQ ID NO: 116:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 116:                         # 20               CGAA                                                       - (2) INFORMATION FOR SEQ ID NO: 117:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 117:                         # 20               ACCA                                                       - (2) INFORMATION FOR SEQ ID NO: 118:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 118:                         # 20               ATCC                                                       - (2) INFORMATION FOR SEQ ID NO: 119:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 119:                         # 20               TTCA                                                       - (2) INFORMATION FOR SEQ ID NO: 120:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 120:                         # 20               CTCC                                                       - (2) INFORMATION FOR SEQ ID NO: 121:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 121:                         # 20               TTCA                                                       - (2) INFORMATION FOR SEQ ID NO: 122:                                         -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (iv) ANTI-SENSE: yes                                                    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 122:                         # 20               GATC                                                       __________________________________________________________________________

What is claimed is:
 1. An oligonucleotide up to 50 nucleotide units inlength comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO:1, 2, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,18, 19, 20, 52 and
 53. 2. The oligonucleotide of claim 1 wherein atleast one of the intersugar linkages between nucleotide units of theoligonucleotide is a phosphorothioate.
 3. The oligonucleotide of claim 1wherein at least one of the nucleotide units comprises a modification onthe 2' position of the sugar.
 4. The oligonucleotide of claim 1 which isa chimeric oligonucleotide.
 5. An isolated nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:105.
 6. The nucleic acidmolecule of claim 5 wherein said nucleic acid molecule isdouble-stranded.
 7. An isolated nucleic acid molecule comprising asequence complementary to the nucleotide sequence of SEQ ID NO:105.
 8. Apolynucleotide probe up to 50 nucleotide units in length comprising anucleotide sequence complementary to a portion of the nucleic acidmolecule of claim 5 or claim
 7. 9. An antisense oligonucleotide 5 to 50nucleotides in length comprising a nucleotide sequence complementary toa portion of the sequence set forth in SEQ ID NO:105.
 10. An antisenseoligonuclcotide 5 to 50 nucleotides in length comprising a nucleotidesequence which is complementary to the long mRNA transcript of humanprotein kinase C-α and which is not complementary to the short mRNAtranscript of human protein kinase C-α.
 11. The anti senseoligonucleotide of claim 10 comprising a nucleotide sequencecomplementary to a portion of the sequence set forth in SEQ ID NO:106.12. The oligonucleotide of claim 11 comprising a sequence as set forthin SEQ ID NO:
 117. 13. A polynucleotide probe comprising a nuclcotidesequence complementary to the long mRNA transcript of human proteinkinase C-α.
 14. The polynucleotide probe of claim 13 comprising asequence as set forth in SEQ ID NO:
 117. 15. A method of inhibiting theexpression of human protein kinase C-α in vitro comprising contactinghuman cells with a therapeutically effective amount of an antisenseoligonucleotide 5 to 50 nucleotides in length, said antisenseoligonucleotide comprising a nucleotide sequence complementary to aportion of the sequence set forth in SEQ ID NO:
 105. 16. A method ofinhibiting the expression of human protein kinase C-α in vitrocomprising contacting human cells with a therapeutically effectiveamount of an antisense oligonucleotide 5 to 50 nucleotides in length,said antisense oligonucleotide comprising a nucleotide sequencecomplementary to a portion of the sequence set forth in SEQ ID NO: 106.17. An oligonucleotide up to 50 nucleotide units in length comprising anucleotide sequence selected from the group consisting of SEQ ID NO:3, 6and
 17. 18. The oligonucleotide of claim 17 wherein at least one of thenucleotide units comprises a modification on the 2' position of thesugar.
 19. The oligonucleotide of claim 17 wherein at least one of theintersugar linkages between nucleotide units of the oligonucleotide is aphosphorothioate.
 20. The oligonucleotide of claim 17 which is achimeric oligonucleotide.